Preparation method and application of ophiopogon root selenium polysaccharide

Abstract

The invention discloses preparation of ophiopogon root selenium polysaccharide and application of the ophiopogon root selenium polysaccharide in preparation of anti-colorectal cancer drugs. The method comprises the following steps: weighing ophiopogon root polysaccharide, putting the ophiopogon root polysaccharide into a container, adding HNO3, and stirring to completely dissolve the ophiopogon root polysaccharide; then adding Na2SeO3 to react, cooling to room temperature after the reaction is completed, and further adding Na2CO3 to adjust the pH value of the reaction liquid; finally, the reaction solution is sequentially subjected to dialysis, vacuum concentration and freeze drying, and the ophiopogon root selenium polysaccharide is obtained. The invention also protects the application in preparation of anti-colorectal cancer drugs. The ophiopogon root selenium polysaccharide prepared by the invention remarkably inhibits proliferation of colorectal cancer caco-2 cells in a dose and time dependent manner, has an obvious inhibiting effect on migration ability and invasion ability of the colorectal cancer caco-2 cells, and can promote apoptosis of the colorectal cancer caco-2 cells.

Description

Technical Field

[0001] The present invention belongs to the technical field of medicine, and specifically relates to a preparation method of ophiopogon japonicus selenium polysaccharide and its use in the preparation of anti-colorectal cancer drugs. Background Art

[0002] At present, cancer treatment mainly relies on surgery, radiotherapy and chemotherapy. Due to the often accompanying complications and difficult-to-control complex situations such as cancer cell metastasis during the treatment process, the treatment effect is not ideal. Therefore, finding more effective anti-tumor drugs and treatment methods remains a difficult problem to be solved in the medical and health care industry.

[0003] Selenium (Se) is an essential dietary trace element in the human body, which is inseparable from human health and plays an important role in anti-tumor, antioxidant, immune regulation, blood sugar lowering and other aspects. Selenium exists in nature in inorganic and active organic forms. Inorganic selenium usually exists in the form of selenate and selenite. Ingesting inorganic selenium easily leads to toxicity accumulation and even mutations. Organic selenium mainly exists in selenoproteins and selenium polysaccharides in animals and plants. Organic selenium has higher biological activity and utilization rate, is more easily absorbed by the human gastrointestinal tract, and shows a higher dose threshold. Among various organic selenium, due to the similar biological functions of selenium and polysaccharides, the combination of selenium and polysaccharides will produce greater biological effects, which is beneficial to human absorption and utilization, and has less side effects. Therefore, the research on selenium polysaccharides has attracted much attention from researchers. However, the sources of natural selenium polysaccharides are very limited, so currently selenium polysaccharides are mainly obtained through artificial synthesis. Ophiopogon japonicus polysaccharide is one of the main active components of ophiopogon japonicus and has various biological activities such as blood sugar lowering, anti-hypoxia, anti-radiation and immune regulation. Existing research has shown that the combination of polysaccharides and selenium can exert the functional activities of selenium and polysaccharides, and can coordinate and enhance the effects of polysaccharides and selenium, thereby effectively improving the activities of polysaccharides and selenium. However, there is currently no relevant report on the combination of ophiopogon japonicus polysaccharide and selenium for the preparation of anti-colorectal cancer drugs. Summary of the Invention

[0004] In order to solve the problems existing in the background art, the present invention uses sodium selenite as a modifier to prepare ophiopogon japonicus selenium polysaccharide in a system with nitric acid as a catalyst, removes the barium chloride catalyst used in the previous preparation of selenium polysaccharide, simplifies the preparation steps, and reduces the use of the dangerous drug barium chloride;

[0005] The ophiopogon japonicus selenium polysaccharide prepared by the present invention can inhibit the proliferation activity, migration ability and invasion ability of colorectal cancer caco-2 cells, and can promote the apoptosis of colorectal cancer caco-2 cells.

[0006] The technical solution of the present invention is as follows:

[0007] I. A preparation method of ophiopogon japonicus selenium polysaccharide, comprising the following steps:

[0008] First, weigh the Ophiopogon japonicus polysaccharide and place it in a container, then add HNO 3 solution, and stir to completely dissolve the Ophiopogon japonicus polysaccharide; then add Na 2 SeO 3 for reaction. After the reaction is completed, cool it to room temperature, and then further add Na 2 CO 3 to adjust the pH of the reaction solution; finally, subject the reaction solution to dialysis, vacuum concentration, and freeze-drying in sequence to obtain Ophiopogon japonicus selenium polysaccharide.

[0009] The mass-to-volume ratio of the Ophiopogon japonicus polysaccharide and the HNO 3 solution is 1 g: 70 - 100 mL.

[0010] In the method, Na 2 SeO 3 is added in a mass ratio of 1: 0.4 - 0.8 based on the Ophiopogon japonicus polysaccharide. 2 SeO 3 .

[0011] After adding Na 2 SeO 3 react at a temperature of 65 - 80 °C for 6 - 7 h.

[0012] In the method, Na 2 CO 3 is added to adjust the pH of the reaction solution to make the reaction solution reach a neutral pH = 7.

[0013] The dialysis mentioned above is carried out using a dialysis bag with MW: 3500 for 48 - 72 h until the dialysis fluid shows no red color detected by ascorbic acid.

[0014] The Ophiopogon japonicus selenium polysaccharide contains O - Se - O bonds and Se = O bonds.

[0015] II. Use of Ophiopogon japonicus selenium polysaccharide: Use in the preparation of anti-cancer drugs. Especially use in the preparation of anti-colorectal cancer drugs.

[0016] The anti-colorectal cancer drug mentioned above inhibits the proliferation of colorectal cancer cells, inhibits the migration and invasion ability of colorectal cancer cells, and promotes apoptosis of colorectal cancer cells.

[0017] It further includes the preparation of Ophiopogon japonicus selenium polysaccharide and its application in the preparation of anti-colorectal cancer drugs.

[0018] The Ophiopogon japonicus selenium polysaccharide prepared by the present invention significantly inhibits the proliferation of colorectal cancer caco-2 cells in a dose- and time-dependent manner, has an obvious inhibitory effect on the migration and invasion ability of colorectal cancer caco-2 cells, and can promote apoptosis of colorectal cancer caco-2 cells.

[0019] The present invention has the following beneficial effects:

[0020] The present invention prepares Ophiopogon japonicus selenium polysaccharide by using nitric acid as a catalyst. Under this condition, the selenium content of the prepared Ophiopogon japonicus selenium polysaccharide reaches 9.749 mg / g. It is verified that the Ophiopogon japonicus selenium polysaccharide of the present invention can significantly inhibit the proliferation activity, migration ability and invasion ability of colorectal cancer caco-2 cells, and significantly promote the apoptosis of colorectal cancer caco-2 cells.

[0021] In summary, Ophiopogon japonicus selenium polysaccharide can inhibit the proliferation activity, migration ability and invasion ability of colorectal cancer caco-2 cells, and can promote the apoptosis of colorectal cancer caco-2 cells. Therefore, it can be further developed as an anti-colorectal cancer drug. Description of the Drawings

[0022] Figure 1 A is the morphology of freeze-dried Ophiopogon japonicus polysaccharide, Figure 1 B is the morphology diagram of freeze-dried Ophiopogon japonicus selenium polysaccharide after selenization modification.

[0023] Figure 2 are the infrared spectrum analysis diagrams of Ophiopogon japonicus polysaccharide (OJS) and Ophiopogon japonicus selenium polysaccharide (OJSS).

[0024] Figure 3 are the scanning electron microscope diagrams of Ophiopogon japonicus polysaccharide (OJS) and Ophiopogon japonicus selenium polysaccharide (OJSS), where A1 and A2 are the scanning electron microscope diagrams of Ophiopogon japonicus polysaccharide, and B1 and B2 are the scanning electron microscope diagrams of Ophiopogon japonicus selenium polysaccharide.

[0025] Figure 4 is the thermogravimetric analysis TGA diagram of Ophiopogon japonicus polysaccharide (OJS) and Ophiopogon japonicus selenium polysaccharide (OJSS).

[0026] Figure 5 is the inhibitory reaction of the proliferation of colorectal cancer caco-2 cells after treatment with Ophiopogon japonicus selenium polysaccharide (OJSS) and Ophiopogon japonicus polysaccharide (OJS), where Figure 5 A is the inhibition rate of Ophiopogon japonicus selenium polysaccharide on the proliferation of colorectal cancer caco-2 cells, Figure 5 B is the inhibition rate of different dose concentrations of Ophiopogon japonicus selenium polysaccharide treated for 24 h and 48 h; Figure 5 C is the inhibition rate of different dose concentrations of Ophiopogon japonicus polysaccharide treated for 24 h and 48 h. Compared with the untreated control group, ** is p < 0.01.

[0027] Figure 6 is the inhibitory reaction of the migration ability and invasion ability of colorectal cancer caco-2 cells after treatment with Ophiopogon japonicus selenium polysaccharide (OJSS), where Figure 6A is the inhibition rate of Ophiopogon japonicus selenium polysaccharide on the migration ability of colorectal cancer caco-2 cells, Figure 6 B is the inhibition rate of Ophiopogon japonicus selenium polysaccharide on the invasion ability of colorectal cancer cells. Compared with the untreated cell group, ** indicates p < 0.01.

[0028] Figure 7 shows the cell cycle arrest of colorectal cancer caco-2 cells after treatment with Ophiopogon japonicus selenium polysaccharide (OJSS). Among them, Figure 7 A is the cell cycle distribution of colorectal cancer caco-2 cells after treatment with Ophiopogon japonicus selenium polysaccharide, Figure 7 B is the percentage of the number of cells in different stages of the cell cycle after treatment with Ophiopogon japonicus selenium polysaccharide. * indicates p < 0.05, and ** indicates p < 0.01.

[0029] Figure 8 shows the apoptosis of colorectal cancer caco-2 cells after treatment with Ophiopogon japonicus selenium polysaccharide (OJSS). Among them, Figure 8 A is the distribution of apoptotic cells in four quadrants after AnnexinV-PI double staining, Figure 8 B is the proportion of early apoptotic and late apoptotic cells of colorectal cancer caco-2 cells. * indicates p < 0.05, and ** indicates p < 0.01.

[0030] Figure 9 shows the expression levels of some genes related to the mitochondrial apoptosis pathway in colorectal cancer caco-2 cells after treatment with Ophiopogon japonicus selenium polysaccharide (OJSS). Compared with the untreated cell group, * indicates p < 0.05, and ** indicates p < 0.01. Specific implementation manners

[0031] The following examples are further illustrations of the present invention, rather than limitations on the present invention.

[0032] Examples of the present invention are as follows:

[0033] 1 Materials and methods

[0034] 1.1 Experimental materials

[0035] Ophiopogon japonicus was purchased from Yibainian Chinese Herbal Medicine Co., Ltd. in Santai County, sodium selenite pentahydrate was purchased from Macklin Biochemical Technology Co., Ltd., nitric acid and sulfuric acid were purchased from Chengdu Kelong Chemical Co., Ltd., gel was purchased from Nanjing Dulai Biotechnology Co., Ltd., cellulose was purchased from Beijing Ruidahenghui Technology Development Co., Ltd., dialysis bags were purchased from Chengdu Kaicheng Technology Co., Ltd., human colorectal cancer caco-2 cells were derived from the Shanghai Institute of Cell Biology, Chinese Academy of Sciences, Annexin V-FITC apoptosis detection kit, BeyoRT II cDNA synthesis kit, and Cell Counting Kit-8 (CCK-8 kit) were purchased from Shanghai Beyotime Biotechnology Co., Ltd., and the cell cycle detection kit was purchased from Beijing Merida Technology Co., Ltd.

[0036] 1.2 Extraction of Ophiopogon japonicus polysaccharide

[0037] The extraction method of Ophiopogon japonicus polysaccharide was the water extraction and alcohol precipitation method. The dried roots of Ophiopogon japonicus were crushed and passed through a 60-mesh sieve. 10 g of dried Ophiopogon japonicus powder was weighed, 100 mL of distilled water was added and stirred evenly. After soaking for 5 h, it was placed in a constant temperature water bath, extracted at 90 °C for 2 h and then filtered to obtain the filtrate. It was concentrated under reduced pressure at 55 °C, precipitated overnight with four times the amount of 95% ethanol, centrifuged, and freeze-dried under vacuum to obtain crude Ophiopogon japonicus polysaccharide. The crude Ophiopogon japonicus polysaccharide obtained by extraction was purified by DEAE-cellulose52 ion exchange column chromatography and Sephadex G-150 gel chromatography column chromatography, and the eluent was distilled water. The purified Ophiopogon japonicus polysaccharide was dialyzed using dialysis bags, freeze-dried, and finally purified Ophiopogon japonicus polysaccharide was obtained, with the morphology as Figure 1 shown in A.

[0038] 1.3 Synthesis and characterization analysis of Ophiopogon japonicus selenium polysaccharide

[0039] 1.3.1 Synthesis of Ophiopogon japonicus selenium polysaccharide

[0040] Weigh 1 g of Ophiopogon japonicus polysaccharide into a 250 mL round-bottom flask, add 1% HNO 3 100 ml, heat and stir to dissolve it completely. According to the mass ratio of Ophiopogon japonicus polysaccharide to Na 2 SeO 3 of 1:0.6, add Na 2 SeO 3 , the reaction temperature is 70 °C, the reaction time is 7 h. After the reaction is completed, cool it to room temperature, and adjust the reaction solution to pH = 7 with 1 mol / L Na 2 CO 3 . The supernatant was dialyzed using dialysis bags (take a small amount of dialysis solution and detect it with ascorbic acid until the detection solution has no red color), concentrated under reduced pressure, and freeze-dried to obtain Ophiopogon japonicus selenium polysaccharide, with the morphology as Figure 1As shown in Figure B. Under these process conditions, the selenium content of the Ophiopogon japonicus selenium polysaccharide obtained was 9.749 mg / g.

[0041] 1.3.2 Fourier transform infrared spectroscopy scanning

[0042] Weigh 2 mg of the Ophiopogon japonicus polysaccharide and Ophiopogon japonicus selenium polysaccharide samples respectively, mix and grind them with potassium bromide (KBr) dried at 105 °C, and press them into thin slices, and then scan them with an infrared spectrometer in the range of 4000 - 400 cm -1 for infrared spectrum scanning.

[0043] 1.3.3 Scanning electron microscopy analysis

[0044] Weigh 10 mg of the Ophiopogon japonicus polysaccharide and Ophiopogon japonicus selenium polysaccharide to be tested respectively, place the samples on the sample stage, spray gold to coat the conductive layer, and scan them with a scanning electron microscope.

[0045] 1.3.4 Thermogravimetric analysis

[0046] Use a thermogravimetric analyzer. Load 3 mg of Ophiopogon japonicus polysaccharide and Ophiopogon japonicus selenium polysaccharide each into an alumina crucible, and under a nitrogen atmosphere with a flow rate of 30 ml·min -1 and a programmed temperature increase of 10 °C·min -1 , in the temperature range of 25 - 1000 °C, conduct the measurement.

[0047] 1.4 Cell culture conditions

[0048] The cells are from the Shanghai Institute of Cell Biology, Chinese Academy of Sciences. They are inoculated into DMEM medium containing 10% fetal bovine serum and 1% non-essential amino acids, and placed in a 5% carbon dioxide incubator at 37 °C for culture. Cells in good growth and in the logarithmic growth phase are used for the following experiments.

[0049] 1.5 Cell proliferation assay CCK-8

[0050] Prepare a cell suspension of 2×10 4 cells / mL from the cells in the logarithmic growth phase with cell culture medium. Inoculate 100 μL per well into a 96-well plate, and culture overnight in a 5% CO2 incubator at 37 °C. After the cells are completely adherent, discard the supernatant. Then add 100 μL of the prepared intervention substances with different concentration gradients (the intervention substances are polysaccharides and selenium polysaccharides) per well. In addition, set up a control group with only cells without adding any test substances and a blank group without cells. At the same time, set 50 μg / mL 5-fluorouracil (5-FU) as the positive control group. Set 6 parallels for each mass concentration, and continue to culture for 24 h and 48 h. Discard the supernatant, add 10 μL of CCK-8 solution and 100 μL of cell culture medium to each well, and after culturing for 2 h, measure the absorbance value at a wavelength of 450 nm with an enzyme-linked immunosorbent assay reader, paying attention to avoiding light

[18] . Calculate the cell inhibition rate using the inhibition rate formula.

[0051] Inhibiting rate=[(Ac - As) / (Ac - Ab)]×100%

[0052] Wherein, Ac——Absorbance of the control well (culture medium containing cells, CCK - 8, without the drug to be tested);

[0053] As——Absorbance of the test well (culture medium containing cells, CCK - 8, with the drug to be tested);

[0054] Ab——Absorbance of the blank well (culture medium without cells and the drug to be tested, CCK - 8).

[0055] 1.6 Cell migration assay

[0056] In this study, the Transwell assay was used to analyze the effect of selenium polysaccharide from Ophiopogon japonicus on the migration of caco - 2 cells. The Transwell chamber was placed in a 24 - well plate. 500 μL of DMEM medium containing 10% fetal bovine serum was added to the lower chamber, and 200 μL of serum - free cell suspension (2×10 4 cells per well) containing selenium polysaccharide from Ophiopogon japonicus (0, 100, 300, 500 μg / mL) was added to the upper chamber. After culturing for 24 h, it was taken out, fixed with 4% paraformaldehyde at room temperature for 15 min, washed twice with PBS, stained with 0.1% crystal violet for 30 min, washed twice with PBS to remove the excess crystal violet, and the inner wall of the chamber was wiped with a moistened cotton swab to remove the non - migrated cells, and then observed and photographed under a microscope.

[0057] 1.7 Cell invasion assay

[0058] The Matrigel was thawed on ice in a 4°C refrigerator. The thawed Matrigel was gently mixed with serum - free medium at a ratio of 1:8. 60 μL of the diluted Matrigel was vertically added to each chamber, avoiding the generation of air bubbles, and then placed in an incubator for 60 min to solidify the Matrigel. After 60 min, the chamber was taken out, the excess liquid was aspirated, 100 μL of serum - free medium was added to each well and then placed back in the incubator for 30 min for Matrigel hydration. After taking out the hydrated chamber, the liquid in the chamber was washed off. 500 μL of DMEM medium containing 10% fetal bovine serum was added to the lower chamber, and 200 μL of serum - free cell suspension (10×10 4 cells per well) containing selenium polysaccharide from Ophiopogon japonicus (0, 100, 300, 500 μg / mL) was added to the upper chamber. After culturing for 48 h, it was taken out, fixed and stained, and then observed and photographed under a microscope.

[0059] 1.8 Flow cytometry analysis of cell cycle

[0060] The caco - 2 cells in the logarithmic growth phase were seeded at 6×10 5Cells were seeded at a density of 6×10 cells / well in a 6-well cell culture plate and cultured in an incubator until they adhered. The original cell culture medium was aspirated, and 1 mL of polysaccharides with different mass concentrations (100, 300, 500 μg / mL) was added to each well. In addition, a control group containing only cells without any test substances was set up. After 24 h of treatment, the following procedures were performed: After washing once with PBS, 0.5 mL of trypsin was added to each well to digest the adherent cells and make the cells into single cells. The cells were collected in a 1.5 mL centrifuge tube and centrifuged at 1000 r / min for 5 min; 1 mL of PBS pre-cooled at 4°C was added to resuspend the cells; after centrifuging again at 1000 r / min for 5 min, 1 mL of 70% ethanol pre-cooled at 4°C was added to fix the cells overnight at 4°C; centrifuged at 1000 r / min for 5 min, the ethanol was discarded, 1 mL of PBS pre-cooled at 4°C was added to wash the cells twice, centrifuged at 1000 r / min for 5 min, and the supernatant was discarded; 0.5 mL of propidium iodide staining solution was added to each tube, incubated at 37°C in the dark for 30 - 60 min, and detected using a flow cytometer with an excitation wavelength of 488 nm.

[0061] 1.9 Flow cytometry analysis of cell apoptosis rate

[0062] Cells were seeded at a density of 6×10 5 Cells were seeded at a density of 6×10 cells / well in a 6-well cell culture plate and cultured in an incubator until they adhered. The original cell culture medium was aspirated, and 1 mL of polysaccharides with different mass concentrations (100, 300, 500 μg / mL) was added to each well. In addition, a control group containing only cells without any test substances was set up. After 24 h of treatment, the following procedures were performed: The supernatant was aspirated into a centrifuge tube, collected in a centrifuge tube after washing once with PBS, 0.5 mL of trypsin was added to each well to digest the adherent cells and make the cells into single cells. The cells were collected in a 15 mL centrifuge tube and centrifuged at 1000 r / min for 5 min; 1 mL of PBS pre-cooled at 4°C was added to wash the cells once; 195 μL of Annexin V-fluorescein isothiocyanate (FITC) binding solution was added to gently resuspend the cells and transferred to a 1.5 mL sterile centrifuge tube; 5 μL of Annexin V-FITC was added, gently mixed, 10 μL of propidium iodide staining solution was added, gently mixed, incubated at room temperature for 25 min, and detected using a flow cytometer.

[0063] 1.10 Detection of the expression of apoptosis-related genes in the mitochondrial pathway by RT-PCR

[0064] Caco-2 cells in the logarithmic growth phase were seeded at a density of 5.3×10 5 cells per well in a six-well plate and placed in an incubator at 37°C with 5% CO 2Cultivate adherent cells in an incubator, discard the culture medium, add 1 mL of polysaccharides with different mass concentrations (100, 300, 500 μg / mL) to each well, and set up a control group with only cells without adding any test substances. After treatment for 24 h, extract cellular RNA using a kit method, measure the RNA concentration with a micro-spectrophotometer, reverse transcribe the RNA into cDNA using a cDNA kit, dilute the cDNA five-fold for quantification, and measure the expression levels of apoptosis-related genes using RT-PCR method with β-actin as an internal reference. The primer sequences were synthesized by Sangon Biotech (Shanghai) Co., Ltd. The RT-PCR reaction conditions were as follows: pre-denaturation at 94 °C for 3 min, denaturation at 95 °C for 10 s, annealing at 60 °C for 30 s, extension at 95 °C for 15 s, for a total of 40 cycles, and relative quantification was performed on each sample using the 2 -△△CT method.

[0065] 2 Experimental result analysis

[0066] 2.1 Structural characterization of Ophiopogon japonicus selenium polysaccharide

[0067] 2.1.1 Fourier transform infrared spectroscopy analysis

[0068] Figure 2 The Fourier transform infrared spectra of Ophiopogon japonicus polysaccharide and Ophiopogon japonicus selenium polysaccharide are shown. The characteristic absorption peaks of Ophiopogon japonicus polysaccharide and Ophiopogon japonicus selenium polysaccharide in the wavenumber range of 4000 - 500 cm -1 are consistent with the literature reports. Ophiopogon japonicus polysaccharide has a strong and broad peak at 3349 cm -1 , attributed to the stretching vibration of O-H. 2938 cm -1 is the stretching vibration peak of C-H, and 1644 cm -1 is the stretching vibration of carboxyl C=O. Absorption peaks in the range of 1100 - 1000 cm -1 indicate the presence of pyranose. However, there are no significant changes in the important characteristic absorption peaks of Ophiopogon japonicus selenium polysaccharide, only changes in the peak shape and absorption wavelength of some functional group characteristic peaks. For example, C-H is red-shifted to 2934 cm -1 , and O-H is slightly blue-shifted to 3377 cm -1 and the peak becomes smaller and weaker. This indicates that due to the substitution of selenite groups, the local spatial position situation has changed, resulting in a change in the state of hydroxyl groups participating in molecular hydrogen bonds, which is beneficial to improving the water solubility of the polysaccharide. The weakening of the peak height indicates that the selenite group has been substituted, reducing the number of hydroxyl groups. In addition, Ophiopogon japonicus selenium polysaccharide shows an absorption peak at 1253 cm -1 , attributed to the O-Se-O bond present in the selenoester, and at 868 cm -1The characteristic absorption peak of the unique selenite group appeared, which was attributed to the Se=O vibration absorption peak. It can be seen that selenium combines with Ophiopogon japonicus polysaccharide in the form of selenite to form Ophiopogon japonicus selenopolysaccharide, and the main structure of Ophiopogon japonicus polysaccharide is not destroyed after selenization.

[0069] 2.1.2 Scanning electron microscopy analysis

[0070] Figure 3 The figure shows the electron microscope scanning images of Ophiopogon japonicus polysaccharide and Ophiopogon japonicus selenopolysaccharide. Refer to the existing SEM analysis of selenopolysaccharide. The surface of Ophiopogon japonicus polysaccharide is irregular geometric block and has a porous structure. The surface of Ophiopogon japonicus selenopolysaccharide has undergone a significant change, forming a smooth and rounded strip structure. When dissolved, it was found that the solubility of Ophiopogon japonicus selenopolysaccharide was better than that of Ophiopogon japonicus polysaccharide, which indicates that after Ophiopogon japonicus polysaccharide is selenized, the aggregation state of Ophiopogon japonicus polysaccharide molecules has changed, and the surface dispersion is better.

[0071] 2.1.3 Thermogravimetric analysis

[0072] Figure 4 The figure shows the thermogravimetric analysis TGA graph of Ophiopogon japonicus polysaccharide and Ophiopogon japonicus selenopolysaccharide. Ophiopogon japonicus polysaccharide begins to decompose at 221.1696℃, at which time the remaining mass of Ophiopogon japonicus polysaccharide is 91.9315% of the total mass of the sample. Ophiopogon japonicus selenopolysaccharide begins to decompose at 170.6156℃, at which time the remaining mass of Ophiopogon japonicus selenopolysaccharide is 87.9315% of the total mass. This indicates that the thermal stability of Ophiopogon japonicus polysaccharide decreases after selenization.

[0073] 2.2 Inhibition of Selenium Polysaccharide from Ophiopogon japonicus on Colorectal Cancer Caco-2 Cells

[0074] 2.2.1 Inhibitory effect of Ophiopogon japonicus selenium polysaccharide on the proliferation of Caco-2 cells

[0075] Figure 5 The figure shows the inhibition rate of Ophiopogon japonicus selenium polysaccharide on the proliferation of caco-2 cells. After 24 hours of treatment with different doses of Ophiopogon japonicus selenium polysaccharide, within the dose range of 50-500 μg / mL, the inhibitory effect of Ophiopogon japonicus selenium polysaccharide on caco-2 cells gradually increased with the increase of dose. And at the same dose concentration, the inhibitory effect of Ophiopogon japonicus selenium polysaccharide on caco-2 cells was time-dependent. However, Figure 5 As shown in C, compared with Ophiopogon japonicus selenopolysaccharide and positive control, Ophiopogon japonicus polysaccharide did not have a significant inhibitory effect on the proliferation of Caco-2 cells, which indicates that Ophiopogon japonicus polysaccharide itself has no inhibitory effect on colorectal cancer Caco-2 cells. After Ophiopogon japonicus polysaccharide was selenized, Ophiopogon japonicus selenopolysaccharide showed an inhibitory effect on the proliferation of Caco-2 cells. This can be further understood as Ophiopogon japonicus polysaccharide has no anti-tumor ability, but the successful selenization modification gives Ophiopogon japonicus polysaccharide anti-tumor ability.

[0076] 2.2.2 Effect of Ophiopogon japonicus selenium polysaccharide on the migration of caco-2 cells

[0077] Figure 6 Figure A shows the microscopic pictures of caco-2 cells and the cell migration rate after treatment with Ophiopogon japonicus selenium polysaccharide. Compared with the control group, the migration rate of caco-2 cells in the Ophiopogon japonicus selenium polysaccharide treatment group decreased, and within the range of 100 - 500 μg / mL of Ophiopogon japonicus selenium polysaccharide, the migration rate decreased more with the increase of the dose concentration. There were significant differences in the cell migration rate among the treatment groups and the control group (p < 0.01), indicating that Ophiopogon japonicus selenium polysaccharide could inhibit the migration of caco-2 cells.

[0078] 2.2.3 Effect of Ophiopogon japonicus selenium polysaccharide on the invasion ability of caco-2 cells

[0079] Figure 6 Figure B shows the microscopic pictures of caco-2 cells and the cell invasion rate after treatment with Ophiopogon japonicus selenium polysaccharide. Compared with the control group, the invasion rate of caco-2 cells in the Ophiopogon japonicus selenium polysaccharide treatment group decreased significantly, and there were extremely significant differences in the cell invasion rate among the treatment groups and the control group (p < 0.01), indicating that Ophiopogon japonicus selenium polysaccharide could inhibit the invasion of caco-2 cells.

[0080] 2.2.4 Effect of Ophiopogon japonicus selenium polysaccharide on the cell cycle arrest of caco-2 cells

[0081] Figure 7 Figure shows the cell cycle arrest of caco-2 cells after treatment with Ophiopogon japonicus selenium polysaccharide. Compared with the untreated group, after treatment with Ophiopogon japonicus selenium polysaccharide, obvious changes occurred in the G0 / G1 phase of caco-2 cells. The concentrations of Ophiopogon japonicus selenium polysaccharide were 100, 300, and 500 μg / mL respectively, and the proportions of G0 / G1 phase cells after treatment were 52.1667%, 56.1000%, and 60.2667% respectively, while the proportion of G0 / G1 cells in the untreated group was 51.1333%. The results showed that Ophiopogon japonicus selenium polysaccharide arrested the caco-2 cell cycle in the G0 / G1 phase, preventing caco-2 cells from dividing, thereby inhibiting the proliferation of caco-2 cells.

[0082] 2.2.5 Effect of Ophiopogon japonicus selenium polysaccharide on the apoptosis of caco-2 cells

[0083] Figure 8The apoptosis of caco-2 cells after treatment with Ophiopogon japonicus selenium polysaccharide is shown. Compared with the untreated group, after treatment with Ophiopogon japonicus selenium polysaccharide, significant changes occurred in the proportions of early apoptotic cells and late apoptotic cells of caco-2 cells. After treating caco-2 cells with Ophiopogon japonicus selenium polysaccharide at concentrations of 100, 300, and 500 μg / mL respectively, the early apoptosis proportions of the cells were 4.1300%, 6.0200%, and 9.2150% respectively, and the late apoptosis proportions of the cells were 6.0100%, 9.6750%, and 16.1500% respectively. The early apoptosis and late apoptosis proportions of the untreated cells were 2.4550% and 1.8200% respectively. The results indicate that Ophiopogon japonicus selenium polysaccharide effectively promoted the apoptosis of caco-2 cells.

[0084] 2.2.6 Effects of Ophiopogon japonicus selenium polysaccharide on the expression of apoptosis genes in the mitochondrial pathway

[0085] Figure 9 As shown, the mitochondrial pathway is one of the main intrinsic pathways of cell apoptosis. In this study, the effects of Ophiopogon japonicus selenium polysaccharide on the apoptosis of caco-2 cells were detected by RT-PCR method, and the expression levels of Smac, CASP3, CASP9, Apaf1, Bad, Bcl2, Bak, and Bax genes were detected. After treatment with Ophiopogon japonicus selenium polysaccharide, the mRNA expression level of the anti-apoptotic gene Bcl2 in caco-2 cells decreased, while the mRNA expression levels of the pro-apoptotic genes Smac, CASP3, CASP9, Apaf1, Bad, Bak, and Bax in the cells increased. It indicates that after treating caco-2 cells with Ophiopogon japonicus selenium polysaccharide, the mitochondrial pathway participated in the apoptosis of caco-2 cells.

[0086] 2.3 In summary, in the present invention, OJS was selenized by a chemical modification method, and then the successful selenization modification of Ophiopogon japonicus polysaccharide was verified by infrared spectrum scanning, scanning electron microscopy analysis, and thermogravimetric analysis. In cell experiments, after Ophiopogon japonicus selenium polysaccharide treatment, the proliferation, migration, and invasion of Caco-2 cells were inhibited, and the cell cycle was arrested at the G0 / G1 phase. The mitochondrial apoptosis pathway was activated, inducing cell apoptosis. The results of cell experiments confirmed that Ophiopogon japonicus selenium polysaccharide could inhibit the proliferation of Caco-2 colorectal cancer cells in vitro, indicating that Ophiopogon japonicus selenium polysaccharide is suitable for further research and development as a potential anti-tumor drug.

[0087] The above specific embodiments are used to explain and illustrate the present invention, rather than to limit the present invention. Any modifications and changes made within the spirit and scope of the protection of the present invention fall within the protection scope of the present invention.

[0088] The above are only the preferred embodiments of the present invention. Therefore, all equivalent changes or modifications made according to the structures, features and principles described in the scope of the present invention patent application are included in the scope of the present invention patent application.

Claims

1. A method for preparing selenium polysaccharide from Radix Ophiopogonis, characterized in that: The following steps are involved: Firstly, weigh Ophiopogon japonicus polysaccharide and put it in a container, then add HNO3 solution and stir until the Ophiopogon japonicus polysaccharide is completely dissolved; Then, Na2SeO3 was added to react, and after the reaction was completed, the solution was cooled to room temperature, and Na2CO3 was further added to adjust the pH of the reaction solution; Finally, the reaction solution was dialyzed, concentrated under reduced pressure, and freeze-dried to obtain Ophiopogon japonicus selenium polysaccharide.

2. The method for preparing selenium polysaccharide from Radix Ophiopogonis according to claim 1, characterized in that: The mass and volume ratio of the Ophiopogon japonicus polysaccharide and the HNO3 solution is 1g:70-100mL.

3. The method for preparing selenium polysaccharide from Radix Ophiopogonis according to claim 1, characterized in that: In the method, Na2SeO3 is added according to the mass ratio of Ophiopogon japonicus polysaccharide to Na2SeO3 of 1:0.4-0.

8.

4. The method for preparing selenium polysaccharide from Radix Ophiopogonis according to claim 1, characterized in that: After adding Na2SeO3, react at 65-80℃ for 6-7h.

5. The method for preparing selenium polysaccharide from Radix Ophiopogonis according to claim 1, characterized in that: In the method, Na2CO3 is added to adjust the pH of the reaction solution so that the reaction solution is neutral.

6. The method for preparing selenium polysaccharide from Radix Ophiopogonis according to claim 1, characterized in that: The dialysis is performed using a dialysis bag with MW: 3500 for 48-72 hours.

7. A selenium polysaccharide from Radix Ophiopogonis, characterized in that: It is prepared according to any one of the preparation methods described in claims 1-6.

8. The use according to claim 7, characterized in that The Ophiopogon japonicus selenium polysaccharide contains O-Se-O bonds and Se=O bonds.

9. The use of selenium polysaccharide from Radix Ophiopogonis according to claim 8, characterized in that: Use in the preparation of anticancer drugs.

10. The use according to claim 9, characterized in that Use in the preparation of anti-colorectal cancer drugs.

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